A number of clinical conditions of vertebrates have sufficiently deleterious effects upon the vertebrate to warrant the administration of some pharmaceutically active agent. Such agents may include (i) vaccines, to protect against diseases such as tetanus, diptheria or whoophing cough, (ii) hormones, e.g. insulin, LHRH, vasopressin, oxytocin, or (iii) drugs, e.g. anti-cancer agents, antibiotics. In these cases, a suitable agent is administered to the vertebrate to invoke immunity, to supplement hormone levels, to eliminate the disease causing agent or to provide a therapeutic effect.
Administration of the pharmaceutical to the vertebrate may be via a number of routes including intramuscular (i.m.), subcutaneous (s.c.), or oral (per os, p.o.) administration. I.m. or s.c. administration of the pharmaceutical suffers from the disadvantages that: relatively specialized skills are required to administer the pharmaceutical; large scale administration may be difficult to perform; it is expensive; and a number of side reactions can occur to the agent being administered. For these reasons oral administration of the pharmaceutical is generally preferred. Many antibiotics (tetracycline, penicillin etc), and hormones (progesterone, oestrogen etc) can be successfully administered via the oral route. There are however drugs, hormones and immunogens whose efficacy is almost totally lost upon oral administration (including Calcitonin, Erythropoetin, Granulocyte Colony Stimulating Factor, Stem Cell Factor, Granulocyte Colony Stimulating Factor, LHRH analogues, Somatostatin, Insulin, Interferons, Plasminogen Activator Inhibitors and species of DNA and RNA). This loss of efficacy may be due either to the inability of the intestinal mucosa to absorb these compounds or the breakdown of these substances by various physiological agents in the intestinal milieu. To some extent this effect can be overcome by the administration of extremely large doses of the pharmaceutical agent. This approach, however, is not economically feasible for many pharmaceutical agents.
In an attempt to overcome the problem of degradation a number of encapsulation methods have been employed which enable the encapsulated material to by-pass both the gastric acidity and the pepsin mediated proteolysis encountered within the lumen of the stomach. Typically these methods have involved enteric coated capsules, which only release their contents upon contact with the higher pH of the upper duodenum and jejunum. While this has greatly increased the oral efficacy of a number of compounds, still many substances are pharmaceutically inactive upon oral delivery and must be administered parenterally. Noteable examples of such compounds include Calcitonin, Erythropoietin, Granulocyte Colony Stimulating Factor, Stem Cell factor, Granulocyte Macrophage Colony Stimulating Factor, Somatostatin, Insulin, LHRH and its analogues, Interferons, Plasminogen Activator Factor, species of DNA and RNA, and many vaccines.
In a further extension of the encapsulation process, several new forms of encapsulation have been designed in recent years with the specific purpose of trapping large quantities of pharmaceuticals in subcellular capsules, in the hope that once protected from the intestinal milieu, the capsules would themselves be taken up from the intestine and release their contents systemically. Two basic forms of these capsules have been developed, nanocapsules (or microcapsules) and nanospheres (or microspheres). In essence these particles can be formed by one of a number of methods, several of which are outlined below:
(i) Solvent Evaporation
In which a compound which is soluble in one solvent is dispersed into a non-miscible solvent and the first solvent is evaporated off. Particles formed in this fashion have been used to administer (parenterally) a number of water insoluble compounds. An example of such a system would be the formation of polyalkylcyanoacrylate nanocapsules in which the antifungal agent, griseofulvin is entrapped.
(ii) Desolvation
In this method a compound is contained in a liquid in which it is soluble (the solvent) and a second liquid (which is miscible with the first liquid, but in which the compound is not soluble) is added to the solvent. As more of the second liquid is added the compound becomes desolvated. During the process of desolvation the compound rich phase (the coacervate) contains an enriched amount of compound which is dispersed as microdroplets in the compound deficient phase. At this stage the coalesced material can be chemically crosslinked by a suitable crosslinking agent to form micro or nano-particles.
Nanoparticles of gelatin or BSA can be prepared in this way. Solutions of these proteins are desolvated by the addition of sodium sulfate, or ammonium sulfate solutions. At the point of desolvation there is an increase in turbidity, which time the nanoparticles can be formed by the addition of a suitable cross-linker such as glutaraldehyde or butanedione.
(iii) Complex coacervation
In this procedure two polyelectrolytes having opposite charge are mixed in aqueous medium so that a spontaneous liquid/liquid phase separation occurs. The phenomenon is limited to polymers having a suitable ionic charge density and chain length. Typically these microspheres are formed by the addition of a polyanion such as Gum Arabic, Alginate, or Polyphosphate, to a polycation such as Gelatin.
(iv) Polymer/polymer incompatablity
This procedure is based upon the observation that two chemically different polymers dissolved in a common solvent are usually incompatible. Thus the mixture will tend to form two phases. The insoluble phase can be used to coat core particles to form microcapsules. An example would be the precipitation of ethyl cellulose from cyclohexane by the addition of polyethylene.
(v) Interfacial Polymerization
In this technique, two reactants, each dissolved in a mutually immiscible liquid, diffuse to the interface between the two liquids where they react to form a capsule wall. An example of such capsule formation would occur if a mixture of Sebacoyl chloride dissolved in an oil phase was emulsified into an aqueous phase containing ethylenediamine.
Oppenheim and coworkers (1982) have used the desolvation process (described above) to prepare insulin nanoparticles. These nanoparticles were found to be highly effective when administered intravenously, however a disappointingly small quantity of insulin was delivered to the systemic circulation when these particles were given orally. It would appear, from this work that although it was possible to protect the insulin from degradation in the intestine it was not possible to target the nanoparticles to the intestinal mucosa in such a way as to cause uptake. The lack of a suitable targetting agent has in fact rendered this type of microencapsulation technique to be generally unsuitable for oral delivery of encapsulated agents.
Recent work in part undertaken by one of the current inventors (WO086/06635 and PCT/AU86/00299, the disclosures of which are incorporated herein by reference) has, however, provided such a targetting mechanism. In this work use was made of two natural uptake mechanisms in the gut. The first mechanism utilizes the natural uptake mechanism for Vitamin B.sub.12. During this uptake Vitamin B.sub.12 firstly binds to intrinsic factor (IF) in the upper small intestine. The Vitamin B.sub.12 -IF complex then passes down the small intestine and binds to an IF receptor located on the surface of the ileal epithelium. The whole Vitamin B.sub.12 -IF-receptor complex is then internalized by receptor-mediated endocytosis and some time later the Vitamin B.sub.12 appears in the serum. It has been shown that it is possible to chemically link peptides to Vitamin B.sub.12 in such a manner that does not interfere with its complexing to IF, and to deliver these molecules to the circulation following oral administration. The use of Vitamin B.sub.12 as a carrier for the oral delivery of active substances is described in PCT/AU86/00299.
In the second mechanism, natural mucosal binding proteins were employed to target various haptens and protein molecules to the gastrointestinal mucosa and elicit their uptake. These binding proteins included bacterial adhesins (987P and K99 pili), a viral adhesin (flu virus), a toxin binding subunit (LTB), as well as a number of plant lectins. This class of molecules was termed carrier molecules.
Both the above described mechanisms do however suffer from the disadvantage that the amount of material which could be delivered through either uptake mechanism was directly proportional to the amount of targetting agent which could be taken up. In this regard, the vitamin B.sub.12 uptake mechanism is limited by the absolute quantity of Vitamin B.sub.12 which is normally absorbed, which in most animals amounts to only a few micrograms.
Furthermore, in order for either carrier system to work effectively the conjugated material (hormone, peptide or drug) must preferably be able to survive the proteolytic environment of the small intestine and must also contain a suitable site for chemical cross-linkage to the carrier. During the conjugation, care must be taken to preserve the pharmacological activity of the active agent both during the conjugation as well as in the final complex. Furthermore, a number of peptides may not be suitable for oral delivery (due to sensitivity to proteolysis, or due to lack of suitable functional groups for conjugation) and so new analogues may need to be developed which possess an appropriate conjugation site or have been designed to resist proteolytic degradation. In this respect the present invention can be distinguished from the previous inventions described above in that the carrier molecule of the present invention is not covalently conjugated to the pharmaceutically active agent, but rather the carrier molecule is either covalently linked to the material/polymer comprising the microsphere, or is associated hydrophobically with the surface of the microsphere during its formation.
Surprisingly, the present inventors have discovered that it is possible to prepare complexes comprising at least one carrier molecule and at least one microparticle comprising an active pharmaceutical agent. More surprisingly, the present inventors have discovered that the carrier in such complexes can enable the complex comprising a relatively large microparticle to be transported to the circulatory or lymphatic drainage system via the mucosal epithelium of a host. Thus, the present invention overcomes the above-described disadvantages of the methods of oral delivery of the prior art, since in the complexes of the present invention the active agent is not chemically modified and its physiological activity is preserved while the microparticle provides a protection against degradation or modification in the gastro-intestinal environment. Furthermore, the microparticles of the invention are linked to a carrier molecule which can specifically target the microparticles to the intestinal epithelium and provoke uptake.
Other advantages of the present invention will be apparent from the objects of the invention and the disclosure of the invention hereinbelow.